Reagents and Methods for Appending Functional Groups to Proteins

a functional group and protein technology, applied in the field of proteins and functional groups appending, can solve the problems of protein stability, protein stability is limited, and the fabrication of protein microarrays is more arduous

Active Publication Date: 2008-01-24
WISCONSIN ALUMNI RES FOUND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0025]In additional embodiments, L is a polymeric linker carrying polyethylene glycol chains, such as those that are comprised in Pegylation reagents, for example, polymethacrylates with polyethylene glycol chains such as those that are commercially available under the trade name POLY PEG (Trademark, Warwick Effect Polymers, UK). The average number of repeating units of a polymer is determined by measuring an average molecular weight of the polymer and dividing that average molecular weight by the weight of a repeating unit. Most typically the number average molecular weight is used to determine average number of repeating units. But as is well known in the art, average molecular weights of polymers can be measured or calculated in several different ways and will typically differ from one another as will the average number of repeating units calculated from them, because a given polymer is not monodisperse.

Problems solved by technology

While DNA microarrays [55] have been produced in a large scale to study gene function, the fabrication of protein microarrays is more arduous.
A major complicating factor is the tremendous chemical complexity of proteins.
In addition, proteins have limited stability and are susceptible to loss of activity when subjected to chemical modification.
Accordingly, one of the major challenges in protein microarray technology is the development of general and facile strategies for protein immobilization.
Although thiols are potent nucleophiles for thioesters, the resultant thioesters are inherently unstable to hydrolysis [9], making the simple transthioesterification of an intein-derived thioester unsuitable for the chemical modification of proteins.
This method, although site-specific, is labor intensive and low yielding.

Method used

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  • Reagents and Methods for Appending Functional Groups to Proteins
  • Reagents and Methods for Appending Functional Groups to Proteins
  • Reagents and Methods for Appending Functional Groups to Proteins

Examples

Experimental program
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example 1

Identification of the Optimal Nucleophile for Thioesters

[0107]To identify the optimal nitrogen nucleophile for a thioester, kinetic studies were performed on a model chromogenic thioester: AcGlySC6H4-p-NO2. The rate of release of the thiophenolate anion was monitored by measuring the change in absorbance at 410 nm (Scheme 1). Nitrogen nucleophiles with conjugate-acid pKa values ranging from 4.6 to 10.6 (Table 1) were used in the experiments. The logarithmic values of the second-order rate constants (k2) of the unprotonated primary amines were plotted against the pKa values of their conjugate acids to yield the Brønsted plot shown in FIG. 2A. The data were fitted to the equation[26]:

log k2=log(AB)+(β+β′)pKa−log(A10βpKa+B10β′pKa)  (1)

In eq 1, A and B are constants, β′ is the slope of the former part of the Brønsted plot, and β is the slope of the latter part.

[0108]

TABLE 1Nucleophiles used in this studyand the pKa values of their conjugate acids.NucleophilepKaCH3ONH24.60[22]F3CCH2NH25....

example 2

Synthesis of Bifunctional Azides

[0112]After identification of two optimal nucleophiles, we proceeded to synthesize two bifunctional reagents bearing those nucleophiles on one end and an azido group on the other. Azides 1 and 2 are both amides of 1-azido-2-aminoethane. Azide 1 has an α-hydrazino acetamido group, which is a more stable analog of the α-hydrazino acetyl group of C2H5O(O)CCH2NHNH2 (Table 1; FIG. 2); azide 2 has a γ-hydrazino acetamido group and is effectively an alkyl hydrazine.

[0113]Azide 1 was synthesized by the route in Scheme 2. Briefly, Boc-protected 1-azido-2-aminoethane was synthesized from Boc-protected 1-bromo-2-aminoethane. After Boc-deprotection, the amine was coupled to tri-Boc-protected α-hydrazino acetic acid. The Boc groups were removed, and azide 1 was isolated as a free base after cation-exchange chromatography with an overall yield of 72%.

[0114]Azide 2 was synthesized by the route in Scheme 3. Briefly, 4-pentenoic acid was subjected to ozonolysis, and t...

example 3

Kinetics of Thioester Cleavage

[0116]Kinetic studies were performed by reacting azides 1 and 2 with a model chromogenic thioester (Scheme 1).

[0117]The rate constants (k2 and k1) for azide 1 were found to be indistinguishable from those of the α-hydrazino acetyl group. The rate constants for azide 2 were, surprisingly, much lower than those of methylhydrazine. This result is contrary to our finding that methyl hydrazine is a somewhat better nucleophile than the α-hydrazino acetyl functional group (FIG. 2). The intrinsic instability of azide 2 is likely to be responsible for this apparent decrease in reactivity.

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Abstract

Methods and reagents for site-selective functionalization of peptides and proteins. The methods most generally involve the reaction of a thioester with hydrazine. Reagents include bifunctional reagents of formula:
H2N—NH—CH2-M-L-FG
and salts thereof where M is a single bond or a chemical group carrying a non-bonding electron pair, such as —C(O)NR′—, where R′ is H, or an alkyl or aryl group; L is an optional linker group as described above; and FG is a functional group having reactivity that is orthongonal to that of the hydrazine group. FG can, among others, be an azide, alkenyl, alkynyl, nitrile (—CN) or triazole group and is preferably an azide group (—N3). Methods and reagents can, for example, be combined with intein-mediated protein splicing to link proteins or fragments thereof to various chemical species or to a surface. Surface immobilization of proteins via the methods herein results in immobilized proteins which substantially retain biological activity and is thus useful for the generation of peptide or protein microarrays. Kits for functionalization and/or immobilization of peptides and proteins are provided as well as microarrays of peptides, proteins or both.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS[0001]This application takes priority from U.S. provisional application Ser. No. 60 / 820,011, filed Jul. 21, 2006, which is incorporated by reference in its entirety herein.STATEMENT REGARDING GOVERNMENT SUPPORT[0002]This invention was made with United States government support awarded by the following agencies: National Institutes of Health (NIH) GM044783. The United States government has certain rights in this invention.BACKGROUND OF THE INVENTION[0003]The success of genome sequencing has heightened the demand for new means to manipulate proteins. An especially desirable goal is the ability to modify a target protein or peptide at a specific site with a functional group of orthogonal reactivity which can in turn be used for protein modification or immobilization.[0004]Site-specific derivatization of proteins and peptides is useful in a variety of research and therapeutic applications. For example, attachment of reporter molecules (labels or ta...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C40B50/18C07K1/113C07K17/06C40B40/10
CPCC07K1/047C07K1/13C07K1/1077
Inventor RAINES, RONALD T.KALIA, JEET
Owner WISCONSIN ALUMNI RES FOUND
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